1. Field of the Invention
The present invention relates to a recombinant calcium-binding photoprotein having a cysteine residue introduced in the amino-terminal region. It also relates to a conjugate wherein a ligand which binds specifically to a substance to be detected is bound via the cysteine residue, and to a method of detecting substances using the conjugate as a luminescence marker.
2. Related Background Art
The calcium-binding photoproteins emit a flash of light upon specific interaction with calcium, and the known calcium-binding photoproteins currently include aequorin, clytin, obelin, mitrocomin, mnemiopsin and berovin. These photoproteins are highly sensitive to calcium ion, while their luminescences are very high and detected extremely sensitively by commercial detection devices at very low detection limit, at less than 1 picogram. These photoproteins are therefore used for detection and quantification of trace calcium ion and as imaging probes for visualization of dynamic changes in intracellular calcium ion. Because the photoproteins exhibit their luminescence by specifically binding with calcium ion, the background signal which poses a problem for ordinary chemilumine scence is virtually absent and the luminescent reaction itself is instantaneous, going to completion within a few seconds, such that a rapid signal with a favorable S/N ratio is obtained.
Since luminescent reaction systems involve enzyme-mediated luminescent reactions, known as bioluminescence, and consist entirely of biological components, they contain no harmful chemical substances (such as radio isotopes or carcinogenic compounds) and are highly safe. Such photoproteins therefore hold much promise as markers for diagnostic agents and the like.
When using a calcium-binding photoprotein such as aequorin as a marker for immunoassay, for example, the marker must be linked with the substance to be detected. Specifically, the marker photoprotein must be either bound directly to the substance, or it must be indirectly bound thereto through some other substance. Throughout the present specification, a substance used to link a substance to be detected with a photoprotein (marker), either by direct or indirect binding with the substance to be detected, will be referred to as a “ligand”. Such ligands include, for example, biotin, avidin, streptavidin, antigens, antibodies and the like, and will be explained in detail hereunder.
Because of the relatively unstable nature of calcium-binding photoproteins, they are prone to loss of luminescence activity when bound to their ligands. For more precise analytical and diagnostic results, the binding ratio of the photoprotein and its ligand is preferably exactly or nearly 1:1. A rapid and precise diagnosis/detection system may be established by using a ligand-photoprotein conjugate with the ligand and photoprotein bound at a ratio of 1:1.
A representative calcium-binding photoprotein is aequorin, obtained from Aequorea aequorea. Aequorin exists as a complex of the apoprotein portion, apoaequorin, the luminescent substrate, coelenterazine, and molecular oxygen. On binding of calcium to the aequorin molecule, a blue flash of light (maximum wavelength: 465 nm) is produced, and coelenteramide (the oxidized form of coelenterazine) and carbon dioxide are also produced. After the luminescent reaction, the calcium ion may be removed from apoaequorin by a chelating agent such as EDTA, and aequorin may be regenerated by incubation at low temperature in the presence a reducing agent, coelenterazine and molecular oxygen. Analysis of the gene coding for apoaequorin has identified that apoaequorin is composed of 189 amino acids. Its amino acid sequence is listed as SEQ. ID. No.1 of the Sequence Listing. Aequorin has a homologous sequence with the calcium-binding protein calmodulin, and has been reported to comprise three “EF hand motif” domains each comprising a helix-loop-helix structure, for calcium binding (Inouye et al., Proc. Natl. Acad. Sci. USA 82(1985): 3154–3158). Also, the results of X-ray crystallography suggest that the 184th tyrosine residue near the C-terminus is involved in stabilization of the peroxide portion of the luminescent substrate coelenterazine (Head et al., Nature 405(2000):372–376).
Biotin-avidin (or streptavidin) binding is the basis of one of the most commonly used diagnosis/detection systems for immunoassay, and successful biotinylation of aequorin has been reported (Zatta et al., Anal. Biochem. Vol. 194, pp. 185–191, 1991). The biotinylated aequorin produced by Zatta et al. was obtained by biotinylating a free amino group (—NH2) in aequorin. The amino groups suspected of being modified by the method of Zatta et al. are one terminal amino group and amino groups of 15 lysines, but it has not been determined which of the amino groups are biotinylated in what proportion. Also, no method has been established for specifically modifying only one specific amino group. That is to say, high-quality biotinylated aequorin obtained by bonding biotin to aequorin via a free amino group, has not yet been provided.
Genetic analysis of calcium-binding photoproteins has revealed the presence of 3–6 cysteine residues in such molecules. Also X-ray structural analysis of aequorin and obelin has shown that none of the cysteine residues form disulfide bonds, but that all are present in a free state. It is further known that the luminescence activity of aequorin can be easily eliminated by modification with a low molecular chemical modifier, such as N-ethylmaleimide or iodoacetic acid, at the cysteine —SH group. (Shimomura et al., Biochemistry 13(1974):3278–3286).
The present inventors have previously reported luminescence activity by a cysteine-free mutant aequorin having the three cysteine residues in the molecule replaced with serine residues (K. Kurose et al., Proc. Natl. Acad. Sci. USA 86(1989):80–84). In another experiment, cysteine was substituted for the 5th serine, 53rd glutamic acid, 71st methionine and 84th glutamic acid in the apoaequorin of the cysteine-free mutant aequorin, and those new apoaequorins were used to form thyroxine-apoaequorin conjugates by binding thyroxine through cysteine and reconstituted to thyroxine-aequorin conjugates (Lewis et al., Bioconjugate Chem. 11:65–70 & 140–145(2000)). However, this involves substitution of serine for all of the three cysteine residues of wild-type aequorin and modification by introduction of new unique cysteine residue. As yet, no aequorin has been obtained which still exhibits luminescence activity with binding of the ligand to the —SH group of a newly introduced cysteine residue, while keeping the three original cysteine residues of wild-type aequorin.
The method of Lewis, et al., whereby aequorin is regenerated from modified (biotinilated) apoaequorin, produces a relatively low yield compared with the modification (biotinilation) of regenerated aequorin. Due to the nature of the mechanism by which the aequorin is regenerated, regeneration of modified aequorine cannot be applied to wide variety of modified apoaequorins but can be applied to apoaequorins modifed with specific modifying compounds with minimal influence on the regeneration process. Modification of regenerated aequorin, on the other hand, can be applied to any type of ligand molecules and thus the ligand molecules are not limited to a specific one. Another disadvantage of the method of Lewis et al. is that the ligand-aequorin conjugate, ligand-apoaequorin conjugate, unmodified aequorin and unmodified apoaequorin are not separated. Thus, a homogeneous ligand-aequorin conjugate wherein the ligand is bound to aequorin via the —SH group of the introduced cysteine residue, which is suitable for actual analysis, is yet to be produced.